LOW-DENSITY-LIPOPROTEIN SUBFRACTIONS AND [CA2+](I) IN VASCULAR SMOOTH-MUSCLE CELLS

Several studies have established that plasma low density lipoprotein (LDL) consists of various discrete subfractions. Using a variety of techniques (analytical ultracentrifugation, equilibrium density gradient ultracentrifugation, and gradient gel electrophoresis), LDL has been fractionated into a maximum of seven subclasses that differ in particle size, density, and physicochemical composition. Recently, a predominance of smaller denser LDL particles has been associated with an increased risk of coronary artery disease. However, other lipoprotein changes, such as elevated triglycerides and lower HDL cholesterol levels, have been shown in patients with a predominance of the smaller denser LDL subfractions. Thus, it is unclear whether the enhanced atherogenic potential is induced by the LDL subfraction pattern per se or by concomitant lipoprotein changes. Because intracellular free Ca2+ is an important second messenger involved in atherogenesis and regulation of vascular tone, we studied the influence of three LDL subfractions (very light [LDL1], 1.030 to 1.033 g/mL; light [LDL2], 1.033 to 1.040 g/mL; and dense [LDL3], 1.040 to 1.045 g/mL) on [Ca2+]i in vascular smooth muscle cells (VSMCs) cultured from rat aorta. LDL subfractions were isolated by density gradient ultracentrifugation from human EDTA-plasma (n=15). [Ca2+]i was measured by fura 2 fluorescence. Basal [Ca2+]i was 77+/-6 nmol/L. Stimulation of VSMCs with dense LDL3 caused a significantly (P<.05) more pronounced increase (+71+/-13 nmol/L) compared with LDL1 (+38+/-8 nmol/L) and LDL2 (+36+/-9 nmol/L). To further investigate the mechanisms leading to the stimulation of [Ca2+]i by LDL subfractions, we incubated VSMCs with the Ca2+ antagonists nifedipine, diltiazem, and verapamil in concentrations up to 10 mumol/L. The LDL-induced increases in [Ca2+]i remained unaffected. Furthermore, the inhibitor of intracellular Ca2+ mobilization (TMB-8) did not prevent the increases in [Ca2+]i. The effects were not mediated by the LDL receptor because [Ca2+]i was equally stimulated by LDL subfractions in LDL-receptorless fibroblasts isolated from patients with familial hypercholesterolemia. Our results indicate that the LDL subfraction with the highest density, LDL3, has a more pronounced effect on [Ca2+]i in VSMCs, which is an important second messenger involved in atherogenesis. These results suggest a possible mechanism that may contribute to the enhanced biological activity of dense LDL3 per se.